Engineered CRISPR-Cas9 Nucleases

Patent metadata

Field	Value
Publication number	US-2019071657-A1
Application number	US-201816155808-A
Country	US
Kind code	A1
Filing date	Oct 9, 2018
Priority date	Aug 28, 2015
Publication date	Mar 7, 2019
Grant date	—

Abstract

Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genomic engineering, epigenomic engineering, genome targeting, and genome editing.

First claim

What is claimed is:

1 . An isolated Streptococcus pyogenes Cas9 (SpCas9) protein, with mutations at one or both of Q695 and Q926, and optionally one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.

2 . The isolated protein of claim 1 , comprising all four of the following mutations:
N497A, R661A, Q695A, and Q926A.

3 . The isolated protein of claim 1 , comprising mutations at one or both of Q695 and Q926, and optionally one, two, three, four, or all five of L169, Y450, N497, R661, and D1135.

4 . The isolated protein of claim 2 further comprising mutations at one, two, three, four, or all five of L169, Y450, N497, R661, and D1135.

5 . The isolated protein of claim 1 , further comprising one or more of the following mutations: D1135E; D1135V; D1135V/R1335Q/T1337R (VQR variant); D1135E/R1335Q/T1337R (EQR variant); D1135V/G1218R/R1335Q/T1337R (VRQR variant); or D1135V/G1218R/R1335E/T1337R (VRER variant).

6 . The isolated protein of claim 1 , further comprising one or more mutations that decrease nuclease activity selected from the group consisting of mutations at D10, E762, D839, H983, or D986; and at H840 or N863.

7 . The isolated protein of claim 6 , wherein the mutations that decrease nuclease activity are:
(i) D10A or D10N, and (ii) H840A, H840N, or H840Y.

8 . A fusion protein comprising the isolated protein of claim 1 , fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.

9 . The fusion protein of claim 8 , wherein the heterologous functional domain is a transcriptional activation domain.

10 . The fusion protein of claim 9 , wherein the transcriptional activation domain is from VP64 or NF- κB p 65.

11 . The fusion protein of claim 10 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.

12 . The fusion protein of claim 11 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

13 . The fusion protein of claim 11 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

14 . The fusion protein of claim 11 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

15 . The fusion protein of claim 14 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein.

16 . The fusion protein of claim 15 , wherein the TET protein is TET1.

17 . The fusion protein of claim 11 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

18 . The fusion protein of claim 17 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase.

19 . The fusion protein of claim 11 , wherein the heterologous functional domain is a biological tether.

20 . The fusion protein of claim 19 , wherein the biological tether is MS2, Csy4 or lambda N protein.

21 . The fusion protein of claim 19 , wherein the heterologous functional domain is FokI.

22 . An isolated nucleic acid encoding the protein of claim 1 .

23 . A vector comprising the isolated nucleic acid of claim 22 .

24 . A host cell comprising the nucleic acid of claim 23 .

25 . A method of altering the genome of a cell, the method comprising expressing in the cell or contacting the cell with the isolated protein of claim 1 , and a guide RNA having a region complementary to a selected portion of the genome of the cell.

26 . The method of claim 25 , wherein the isolated protein or fusion protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.

27 . The method of claims 25 , wherein the cell is a stem cell; is in a living animal; or is in an embryo.

28 . The method of claims 27 , wherein the stem cell is an embryonic stem cell, mesenchymal stem cell, or induced pluripotent stem cell.

29 . A method of altering a double stranded DNA D (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the isolated protein of claim 1 , and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.

30 . The method of claim 29 , wherein the dsDNA molecule is in vitro.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Kleinstiver Benjamin
• Pattanayak Vikram

Classifications

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title